Arrestins play an essential role in regulating signaling and trafficking of G protein-coupled receptors (GPCRs). Visual arrestins (arrestin-1 and 4) specifically interact with opsins while non-visual arrestins (arrestin-2 and 3) interact with multiple GPCRs and also bind phosphoinositides, components of the endocytic machinery (e.g., clathrin and 22-adaptin), and various signaling molecules (e.g., Src family members and MAP kinases). Crystallographic analysis reveals that arrestins have a similar basal structure containing N- and C-domains joined by a polar core. Disruption of the polar core is thought to occur upon receptor binding and result in an "active" conformation that regulates arrestin interaction with GPCRs and additional proteins. Although the structural basis for arrestin activation as well as for arrestin interaction with GPCRs and signaling molecules remains largely unknown, we recently co-crystallized arrestin-2 with phosphoinositides and with clathrin, key components of the endocytic machinery. In this application, we propose to extend our structural analysis of arrestin-2 and further define its role in binding receptors and mediating endocytosis. The specific objectives are to: 1. Perform crystallographic analysis of arrestin complexes with 22-adaptin and a phosphorylated receptor peptide. Although structure/function analysis has provided important insight into the domains that mediate arrestin interaction with receptors and 22-adaptin, mechanistic insight into the integration of these binding events is lacking. We propose to crystallize and solve structures of arrestin-2 in complex with a phosphorylated receptor peptide and 22-adaptin. High-resolution crystallographic analysis of these complexes should provide insight into: 1) the binding interfaces of arrestin-2 with receptor and 22-adaptin; 2) the mechanism of arrestin-2 activation; and 3) the mechanism of assembly of a multi-protein endocytic complex. 2. Characterize the biology of arrestin interaction with GPCRs and endocytic proteins. The dynamics of arrestin interaction with clathrin-coated pits is complex and involves multiple protein-protein and protein-lipid interactions. We will use insight from our structural analysis to further define the mechanistic basis for arrestin-mediated regulation of GPCR endocytosis. These studies will utilize various biochemical, molecular, and cell biological strategies to probe the interaction of arrestins with endocytic components and elucidate the function of such interactions in receptor trafficking. Overall, these studies should provide insight into differences in the basal and activated forms of arrestin-2 and provide a mechanistic basis for how non-visual arrestins function as GPCR-regulated adaptor proteins. [unreadable] [unreadable] [unreadable]